Recently, an excessive oxidation reaction, what is called oxidative stress, occurring inside cells has been found to exert harmful effects in living bodies. This oxidation reaction is evoked by a molecular, what is called “reactive oxygen species (ROS)” which has a strong oxidative power generated inside cells. Examples of the known ROS include hydrogen peroxide, singlet oxygen, hydroxyl radical, superoxide anion, and the like. In nature, the ROS is used for elimination of a foreign substance in the immune system, detoxification of harmful molecules, or the like, and it also has useful aspects in vivo. In addition, there exists a system of eliminating the ROS by an enzyme or a molecule having an antioxidative capability in cells. Thus, the harmful effects of the ROS are inhibited by rapid removal of excessively generated ROS.
However, when a balance between the generation and the elimination of the ROS is lost so that the generation of the ROS becomes excessive, it initiates to exert the harmful effects in vivo. The oxidative stress damages molecules (such as DNA, protein, or lipid) that are essential for normal activities of cells. As a result, ageing and diseases such as cancer, arteriosclerosis, myocardial infarction, and diabetes can probably be caused. The oxidative stresses induced by unhealthy lifestyle such as excessive exercises, inadequate exercises, unbalanced diet, and smoking. The oxidative stress has become an issue closely related to current life.
Additionally, ROS has been a focus of attention as the pathogenesis of ischemia-reperfusion injury. In ischemia, the organs where the blood flow stopped are placed in the absence of oxygen, and a large amount of the ROS is generated by rapid reoxygenation after the reperfusion. This phenomenon damages a wide range of cells, and probably evokes an injury at an organ level. A risk causing the reperfusion injury makes prompt treatment difficult in the ischemic organs (Droge, W., Physiol. Rev., 82, 47-95, 2002).
Thus, under the situation in which various disadvantageous effects by ROS are discussed, measuring changes in distribution and amount of ROS generated in vivo should give an important insight in respect to a mechanism of disease development and ageing, and should further contribute largely to the development of the effective prophylaxis and treatment. For this purpose, a technique to efficiently measure the behavior of ROS in vivo is required.
An Example of a method of measuring ROS include a method using a chemiluminescent reagent such as isoluminol. This method is to measure a luminescent signal that is accompanied with an oxidation reaction of the reagent with the ROS. This reaction is widely used as a method of evaluating an immunological activity of leukocytes in order to measure ROS released during the attack against a foreign substance that enters a living body (Dahlgren, C. and Karlsson, A., J. Immunol. Meth., 232, 3-14, 1999). Unfortunately, isoluminol has a poor permeability across cell membranes. So, the measurement of ROS released outside cells can be performed, but the method is unsuitable for the measurement of ROS generated inside the cells. In addition, the intensity of this luminescence is weak. Since the method requires a large number of cells for this measurement and a prolonged integration time even for a high-sensitive detector, it is difficult to efficiently measure the ROS.
A measurement using an organic fluorescent dye has been also carried out. The dye, 2′,7′-dichlorodihydrofluorescein, dihydroethidium, and the like are widely used (Munzel, T., et al., Arterioscler. Thromb. Vasc. Biol., 22, 1761-1768, 2002). Although these are nonfluorescent compounds under non-oxidative condition, these compounds become fluorescent by changing their structures due to an oxidation reaction with ROS. This fluorescent intensity indicates to an amount of the ROS in situ. These reagents can be introduced in cells by passing through cell membranes. However, since these reagents cannot be selectively localized in a particular organelle inside the cells, it is difficult to measure the ROS at a particularly localized area. Moreover, the fluorescence emission of these dyes is irreversible, and these cannot repeat the measurement because once the oxidized dye lose the reactivity with the ROS any more.
Recently, a fluorescent protein has been isolated from Aequorea victoria. One improved in its feature is widely used as a probe reagent for measuring a physiological activity in cells. A probe reagent that is composed of proteins can be used as a form of the gene. By being incorporated into cells as a plasmid, the expressed probe reagent can be used for the measurement. This method also allows only a specific cell in a living body or a specific organelle in cells to limitedly express the probe reagent, and makes it possible to measure the physiological activity at a more limited region.
There have been developed oxidative stress probe reagents using fluorescent proteins. Such reagents have a structure in which the fluorescent protein is fused to a marker protein that detects ROS. A protein that changes its structure due to the reaction with the ROS is used as the marker protein. The change in the structure is designed to change the intensity and wavelength of the fluorescence emitted by the fluorescent protein. For example, a probe reagent, what is called “Hyper”, uses a protein reacting specifically with hydrogen peroxide as a sensor protein, and the protein has a structure attached to a fluorescent protein, YFP. The reaction of hydrogen peroxide with the sensor protein results in change in fluorescent intensity of YFP (Belousov, V. V., et al., Nature Methods, 3, 281-286, 2006 Rev., 82, 47-95, 2002). Another example includes an application using fluorescence resonance energy transfer. This probe reagent has a structure in which a sensor protein is sandwiched between two types of fluorescent proteins having different fluorescent wavelength. The change in the structure resulting from the reaction of the sensor protein with the ROS alters the distance between the fluorescent proteins at each end. This change in distance gives a change in the efficiency of the energy transfer. This reagent can monitor ROS by measuring a change in fluorescence spectra (JP Patent Publication (Kokai) No. 2005-95171A). However, the fluorescent change that is based on the structural change of the sensor protein of the reagent is small, and the detection becomes difficult when ROS is slightly produced in particular.
There is a protein that is expressed for a protective response against oxidative stress when cells are exposed to the stress. The oxidative stress of the cells can be detected by measuring an increase in expression of such a protein. In an example that has measured oxidative stress in muscle, proteins such as TNFα and pIκB have been measured as indicators of the oxidative stress (JP Patent Publication (Kokai) No. 2006-162346A). In this method, the intended proteins are measured using a biochemical technique such as Western blotting. Specifically, proteins in cells are extracted by disrupting the cells and then the proteins are subjected to separation. Thus, because the troublesome procedures are required in the prior art, it is impossible to measure the ROS quickly and continuously in living cells